Transcriptional Analysis And Colning Of The Differentially Expressed Protein Genes In Two Ecotypes Of Leymus Chinensis

Leymus chinensis has important ecological significance and also in the development of animal husbandry with significant economic and social benefits. Its clear differentiation of natural populations, and have rich intraspecific genetic type. Yellow-green and gray-green two ecotypes of Leymus chinensis have a significant difference in physiological and ecological characteristics of photosynthesis, resistance, genetic diversity, but the molecular mechanism has not been elucidated. In order to explore the relationship between Leymus chinensis both ecological differences and gene expression, we start from two ecotypes of Leymus chinensis leaves different protein to analysis the expression of these genes in different period and under the NaCl stress. And clone the LcGAPDHgene. The main results were summarized as follows.We design the primer of Leymus chinensis GAPDH,PGK,NBS-LRR and rbcL gene by homologous species conserved region,and analysis of two ecotypes in green period, turning growth period and fruit period by semi-quantitative RT-PCR. The results reveal the gene of GAPDH,which is participate in glycolytic metabolism,and the expression of gray-type Leymus chinensis was higher than yellow-type Leymus chinensis in turning green period,1.87times for other period,and higher1.78bimes than yellow-type.Yellow-type is highest expression in green period,and higher than gray-type for1.39times.PGK and rbcL enzyme participate in Calvin metabolism,the expression of the two genes were similar and both of them were incresased in fruit period,gray-types> yellow-types. NBS-LRR class gene expression gradually increased with the growth of Leymus chinensis and peak in the fruit period.Yellow-type were significantly higher than gray-type in green period and fruit period,namely its1.75times and1.5times.The growth period Leymus chinensis were treated with salt-stress and then analysed by the semi-quantitative RT-PCR. The results showed GAPDH is a adversity response protein and the encoding gene as a reference gene is not appropriate. The expression of LcPGK and LcrbcL genes were inhibited to some extent and the photosynthesis were limited under stress conditions. NBS-LRR gene expression trend to increased with the increasing concentration of salt stress. Based on the conserved sequence,primers were designed for3’RACE and5’RACE and obtain a3’RACE and5’RACE sequence,named LcGAPDH.The cDNA was1463bp in length including a1014bp ORF.The LcGAPDHencoded a protein of337amino acids with a deduced molecular mass of36.5KD and the pI of6.19. It is a hydrophilic proteins and having8glycosylation sites and18phosphorylation sites,secondary structure of the hybrid type.The152-159amino acid region of LcGAPDH have a typical GAPDH protein signaling.The LcGAPDH protein displayed high similarity with Gap C.Speculated GAPDH is the cytoplasm dependent Gapc protein, and it differentially expressed in the two ecotypes of Leymus chinensis mechanism of action remains to be elucidated.